Nitrite reduction by Co(II) and Mn(II) substituted myoglobins: towards understanding necessary components of Mb nitrite reductase activity

Nitrite reduction to nitric oxide by heme proteins is drawing increasing attention as a protective mechanism to hypoxic injury in mammalian physiology. Here we probe the nitrite reductase (NiR) activities of manganese(II)- and cobalt(II)-substituted myoglobins, and compare with data obtained previously for the iron(II) analog wt Mb^II. Both Mn^IIMb and Co^IIMb displayed NiR activity, and it was shown that the kinetics are first order each in [protein], [nitrite], and [H⁺], as previously determined for the Fe^II analog wt Mb^II. The second order rate constants (k₂) at pH 7.4 and T = 25 °C, were 0.0066 and 0.015 M^− 1 s^− 1 for Co^IIMb and Mn^IIMb, respectively, both orders of magnitude slower than the k₂ (6 M^− 1 s^− 1) for wt Mb^II. The final reaction products for Mn^IIMb consisted of a mixture of the nitrosyl Mn^IIMb(NO) and Mn^IIIMb, similar to the products from the analogous NiR reaction by wt Mb. In contrast, the products of NiR by Co^IIMb were found to be the nitrito complex Co^IIIMb(ONO⁻) plus roughly an equivalent of free NO. The differences can be attributed in part to the stronger coordination of inorganic nitrite to Co^IIIMb as reflected in the respective M^IIIMb(ONO⁻) formation constants K_nitrite: 2100 M^− 1 (Co^III) and <~0.4 M^− 1 (Mn^III). We also report the formation constants (3.7 and 30 M^− 1, respectively) for the nitrite complexes of the mutant metmyoglobins H64V Mb^III(NO₂⁻) and H64V/V67R Mb^III(ONO⁻) and a K_nitrite revised value (120 M^− 1) for the nitrite complex of wt metMb. The respective K_nitrite values for the three ferric proteins emphasize the importance of a H-bonding residue, such as His64 in the Mb^III distal pocket or the Arg67 in H64V/V67R Mb^III, in stabilizing nitrite coordination. Notably, the NiR activities of the corresponding ferrous Mbs follow a similar sequence suggesting that nitrite binding to these centers are analogously affected by the H-bonding residues.     

Photochemical and photophysical properties of ruthenium(II) bis-bipyridine bis-nitrile complexes: Photolability

The electrochemical and photophysical properties of two bis-nitrilo ruthenium(II) complexes formulated as [Ru(bpy)₂(L)₂](PF₆)₂, where bpy is 2,2′-bipyridine and L is AN = CH₃CN and sn = NC-CH₂CH₂-CN, have been investigated. Electrochemical data are typical of Ru-bpy complexes with two reversible reduction peaks located near −1.3 and −1.6 V assigned to each bipyridine ligand and one Ru^II/Ru^III oxidation wave centered at approximately +1.5 V. The sn derivative is both IR and Raman active with its coordinated CN stretch appearing at 2277 cm⁻¹ and 2273 cm⁻¹, respectively. The UV/Vis absorption spectrum of the sn derivative is dominated by an intense (ε_max ∼ 58700 M⁻¹ cm⁻¹) absorption band at 287 nm assigned as a LC (π → π∗) transition. The peak observed at 418 nm (ε ∼ 10 400 M⁻¹ cm⁻¹) is an MLCT band while the one at 244 nm (ε ∼ 23 600 M⁻¹ cm⁻¹) is of LMLCT character. The AN derivative behaves similarly. Both complexes show low-temperature emission at around 537 nm with a lifetime near 10.0 μs. ¹H and ¹³C assignments are consistent with the formulation of the complexes. The complexes undergo photosubstitution of solvent with quantum efficiencies near one. Calculated and experimental results support replacement of the nitrile ligands by solvent. Based on DFT calculations, the electron density of the HOMO lies on the metal center, the bipyridine ligands and the nitrile ligands and electron density of the LUMO resides primarily on the bipyridine ligands. The electronic spectra obtained from TDDFT calculations closely match the experimental ones.     

Role of Phe113 at the distal side of the heme domain of an oxygen-sensor (Ec DOS) in the characterization of the heme environment

The heme-based oxygen-sensor enzyme from Escherichia coli (Ec DOS) is a heme-regulated phosphodiesterase with activity on cyclic-di-GMP and is composed of an N-terminal heme-bound sensor domain with the PAS structure and a C-terminal functional domain. The activity of Ec DOS is substantially enhanced by the binding of O₂ to the Fe(II)-protoporphyrin IX complex [Fe(II) complex] in the sensor domain. The binding of O₂ to the Fe(II) complex changes the structure of the sensor domain, and this altered structure becomes a signal that is transduced to the functional domain to trigger catalysis. The first step in intra-molecular signal transduction is the binding of O₂ to the Fe(II) complex, and detailed elucidation of this molecular mechanism is thus worthy of exploration. The X-ray crystal structure reveals that Phe113 is located close to the O₂ molecule bound to the Fe(II) complex in the sensor domain. Here, we found that the O₂ association rate constants (>200 × 10⁻³ μM⁻¹ s⁻¹: F113L; 26 × 10⁻³ μM⁻¹ s⁻¹: F113Y) of the Fe(II) complexes of Phe113 mutants were markedly different from that (51 × 10⁻³ μM⁻¹ s⁻¹) of the wild-type enzyme, and auto-oxidation rates (0.00068 min⁻¹: F113L; 0.039 min⁻¹: F113Y) of the Phe113 mutants also differed greatly from that (0.0062 min⁻¹) of the wild-type enzyme. We thus suggest that Phe113, residing near the O₂ molecule, has a critical role in optimizing the Fe(II)-O₂ complex for effective regulation of catalysis by the oxygen-sensor enzyme. Interactions of CO and cyanide anion with the mutant proteins were also studied.     

Synthesis, structure and DNA binding studies of a mononuclear cobalt(III) complex with a NNO donor Schiff base derived from 4-methyl-2,6-dibenzoylphenol and ethane-1,2-diamine

The mononuclear cationic cobalt(III) complex 1 [Co(L)₂]⁺ [where HL is H₂N(CH₂)₂NC(Ph)C₆H₂(Me)(OH)COPh] has been obtained by the condensation of 4-methyl-2,6-dibenzoylphenol and ethane-1,2-diamine in the presence of CoCl₂ · 6H₂O and isolated as the chloride solvate {1 · Cl · 2H₂O · EtOH}. The water and ethanol do not form part of the cobalt(III) pseudo-octahedral [Co(L)₂]⁺ coordination sphere, which is cis-CoN₄O₂ with the NNO-ligands mer. Compound 1 has been characterised by mass spectrometry, IR, electronic, ¹H and ¹³C NMR spectroscopy, conductivity measurements, elemental analysis, TGA, cyclic voltammetry and an X-ray structural determination. The average Co-N and Co-O distances are, respectively, 1.929(4) and 1.882(3) Å. The N?N intraligand bite distance is 2.73(4) Å and the N-Co-N and O-Co-O angles are, respectively, 86.2(15)° and 93.3(13)°. Cyclic voltammetric studies of complex 1 indicate an irreversible cathodic peak (E_pc, ca. −0.61 V) corresponding to the reduction of cobalt(III) to cobalt(II). Absorption titration experiments gave a binding constant for DNA interaction of 1.4 × 10⁴ M⁻¹ and a binding site size 0.16 base pairs.     

Redox and catalytic properties of alkoxides based on tris(3,5-dimethylpyrazolyl)borato tungsten nitrosyls

Previously we reported on the catalytic properties of species based on the {Mo(NO)(Tp^Me2)O₂} moiety in the cathodic reduction of chloroform. Here, we have performed cyclic voltammetry and spectroscopic studies of the tungsten bis-alkoxide [W(NO)(Tp^Me2)(OEt)₂], a novel chelate [W(NO)(Tp^Me2)O(CH₂)₄O], and a mono-alkoxide [W(NO)(Tp^Me2)Cl(OEt)] [Tp^Me2 = hydrotris(3,5-dimethylpyrazol-1-yl)borate]. All these complexes efficiently catalyse the cathodic reduction of chloroform which proceeds even at ca. −1.77 V versus Fc⁺/Fc in the presence of the chloro(ethoxy) complex. The chelate complex exhibits a quasi-reversible one-electron reduction at a potential 180 mV more anodic than its bis(ethoxy) counterpart. The UV-Vis spectrum of the former complex shows a red-shifted band (by 70 nm) in the visible region when compared with the latter.     

A study on the reaction of copper complex of dioxotetraamine with superoxide ion by spectrophotometry and pulse radiolysis

The reactions of a dioxotetraamine Cu(II) complex [Cu(H₋₂L)] (L is 6-(9-fluorenyl)-1,4,8,11-tetraazaandencane-5,7-dione)with O₂ ⁻ were investigated by electrochemistry, UV-Vis spectrophotometry and pulse radiolysis, respectively. In DMSO solution, [Cu^II(H₋₂L)] was oxidized into [Cu^III(H₋₂L)]⁺ by O₂ ⁻, a consecutive reaction was observed with [Cu^III(H₋₂L)(O₂ ²⁻)] ⁻ as intermediates (k₁=1.71×10³ M⁻¹ s⁻¹, k₂=1.2×10⁻² s⁻¹). The mechanism of O₂ ⁻ dismutation catalyzed by the complex involved alternate oxidation and reduction of Cu(II) by O₂ ⁻ and the k_cat is 6.07 × 10⁷ M⁻¹ s⁻¹ (pH 7.4).     

Reduction of oxaporphyrin ring of CO-bound α-verdoheme complexed with heme oxygenase-1 by NADPH-cytochrome P450 reductase

Heme oxygenase (HO) catalyses the degradation of heme to biliverdin, carbon monoxide (CO) and ferrous iron via three successive monooxygenase reactions, using electrons provided by NADPH-cytochrome P450 reductase (CPR) and oxygen molecules. For cleavage of the oxaporphyrin ring of ferrous α-verdoheme, an intermediate in the HO reaction, involvement of a verdoheme π-neutral radical has been proposed. To explore this hypothetical mechanism, we performed electrochemical reduction of ferrous α-verdoheme-rat HO-1 complex under anaerobic conditions. Upon binding of CO, an O₂ surrogate, the midpoint potential for one-electron reduction of the oxaporphyrin ring of ferrous α-verdoheme was increased from −0.465 to −0.392 V vs the normal hydrogen electrode. Because the latter potential is close to that of the semiquinone/reduced redox couple of FAD in CPR, the one-electron reduction of the oxaporphyrin ring of CO-bound verdoheme complexed with HO-1 is considered to be a thermodynamically likely process. Indeed the one-electron reduced species, [Fe^II(verdoheme•)], was observed spectroscopically in the presence of CO in both NADPH/wild-type and FMN-depleted CPR systems under anaerobic conditions. Under physiological conditions, therefore, it is possible that O₂ initially binds to the ferrous iron of α-verdoheme in its complex with HO-1 and an electron is subsequently transferred from CPR, probably via FAD, to the oxaporphyrin ring.     

Probing hydrogen peroxide oxidation kinetics of wild-type Synechocystis catalase-peroxidase (KatG) and selected variants

Catalase-peroxidases (KatGs) are unique bifunctional heme peroxidases that exhibit peroxidase and substantial catalase activities. Nevertheless, the reaction pathway of hydrogen peroxide dismutation, including the electronic structure of the redox intermediate that actually oxidizes H₂O₂, is not clearly defined. Several mutant proteins with diminished overall catalase but wild-type-like peroxidase activity have been described in the last years. However, understanding of decrease in overall catalatic activity needs discrimination between reduction and oxidation reactions of hydrogen peroxide. Here, by using sequential-mixing stopped-flow spectroscopy, we have investigated the kinetics of the transition of KatG compound I (produced by peroxoacetic acid) to its ferric state by trapping the latter as cyanide complex. Apparent bimolecular rate constants (pH 6.5, 20 °C) for wild-type KatG and the variants Trp122Phe (lacks KatG-typical distal adduct), Asp152Ser (controls substrate access to the heme cavity) and Glu253Gln (channel entrance) are reported to be 1.2 × 10⁴ M^− 1 s^− 1, 30 M^− 1 s^− 1, 3.4 × 10³ M^− 1 s^− 1, and 8.6 × 10³ M^− 1 s^− 1, respectively. These findings are discussed with respect to steady-state kinetic data and proposed reaction mechanism(s) for KatG. Assets and drawbacks of the presented method are discussed.     

Electrochemical assay for the determination of nitric oxide metabolites using copper(II) chlorophyllin modified screen printed electrodes

This work presents a novel electrochemical assay for the collective measurement of nitric oxide (NO) and its metabolites nitrite (NO₂⁻) and nitrate (NO₃⁻) in volume miniaturized sample at low cost using copper(II) chlorophyllin (CuCP) modified sensor electrode. Zinc oxide (ZnO) incorporated screen printed carbon electrode (SPCE) was used as a host matrix for the immobilization of CuCP. The morphological changes of the ZnO and CuCP modified electrodes were investigated using scanning electron microscopy. The electrochemical characterization of CuCP–ZnO–SPCE exhibited the characteristic quasi-reversible redox peaks at the potential +0.06 V versus Ag/AgCl. This biosensor electrode showed a wide linear range of response over NO concentrations from 200 nM to 500 μM with a detection limit of 100 nM and sensitivity of 85.4 nA μM⁻¹. Furthermore, NO₂⁻ measurement showed linearity of 100 nM to 1 mM with a detection limit of 100 nM for NO₂⁻ and sensitivity of 96.4 nA μM⁻¹. Then, the concentration of NO₃⁻ was measured after its enzymatic conversion into NO₂⁻. Using this assay, the concentrations of NO, NO₂⁻, and NO₃⁻ present in human plasma samples before and after beetroot supplement were estimated using suitable membrane coated CuCP–ZnO–SPCE and validated with the standard Griess method.     

The peroxidase and peroxynitrite reductase activity of human erythrocyte peroxiredoxin 2

Peroxiredoxin 2 (Prx2) is a 2-Cys peroxiredoxin extremely abundant in the erythrocyte. The peroxidase activity was studied in a steady-state approach yielding an apparent K_M of 2.4 μM for human thioredoxin and a very low K_M for H₂O₂ (?0.7 μM). Rate constants for the reaction of peroxidatic cysteine with the peroxide substrate, H₂O₂ or peroxynitrite, were determined by competition kinetics, k₂ = 1.0 × 10⁸ and 1.4 × 10⁷ M⁻¹ s⁻¹ at 25 °C and pH 7.4, respectively. Excess of both oxidants inactivated the enzyme by overoxidation and also tyrosine nitration and dityrosine were observed with peroxynitrite treatment. Prx2 associates into decamers (5 homodimers) and we estimated a dissociation constant K_d < 10⁻²³ M⁴ which confirms the enzyme exists as a decamer in vivo. Our kinetic results indicate Prx2 is a key antioxidant enzyme for the erythrocyte and reveal red blood cells as active oxidant scrubbers in the bloodstream.     

The superoxide reductase from the early diverging eukaryote Giardia intestinalis

Unlike superoxide dismutases (SODs), superoxide reductases (SORs) eliminate superoxide anion (O₂^•−) not through its dismutation, but via reduction to hydrogen peroxide (H₂O₂) in the presence of an electron donor. The microaerobic protist Giardia intestinalis, responsible for a common intestinal disease in humans, though lacking SOD and other canonical reactive oxygen species-detoxifying systems, is among the very few eukaryotes encoding a SOR yet identified. In this study, the recombinant SOR from Giardia (SOR_Gi) was purified and characterized by pulse radiolysis and stopped-flow spectrophotometry. The protein, isolated in the reduced state, after oxidation by superoxide or hexachloroiridate(IV), yields a resting species (T_final) with Fe³⁺ ligated to glutamate or hydroxide depending on pH (apparent pK_a = 8.7). Although showing negligible SOD activity, reduced SOR_Gi reacts with O₂^•− with a pH-independent second-order rate constant k₁ = 1.0 × 10⁹ M^− 1 s^− 1 and yields the ferric-(hydro)peroxo intermediate T₁; this in turn rapidly decays to the T_final state with pH-dependent rates, without populating other detectable intermediates. Immunoblotting assays show that SOR_Gi is expressed in the disease-causing trophozoite of Giardia. We propose that the superoxide-scavenging activity of SOR in Giardia may promote the survival of this air-sensitive parasite in the fairly aerobic proximal human small intestine during infection.     

Particle reworking and solute transport by the sediment-living polychaetes Marenzelleria neglecta and Hediste diversicolor

This experimental study quantified and compared particle-mixing and solute transport by the polychaetes Marenzelleria neglecta (2 g ww, 3200 ind. m^− 2) and Hediste diversicolor (2 g ww, 800 ind. m^− 2) in Baltic Sea sediments. Particle tracers (luminophores) were added to the sediment surface and their vertical distribution in the sediment was measured after 10 d. The rate of particle mixing was quantified using a gallery-diffusion model calculating the biodiffusion coefficient D_b and the non-local transport parameter r. Bioirrigation was measured by adding an inert solute tracer (bromide) to the overlying water 1, 1.5 and 2 d before the end of the experiment, and quantified by calculating the net bromide flux and fitting the bromide profiles to a 1D diffusion model providing an apparent biodiffusion coefficient D_a. The two polychaete worms displayed similar particle-mixing and solute transport efficiencies (based on total biomass) despite different modes of bioturbation. However, H. diversicolor was a more efficient particle-reworker and M. neglecta a more efficient bioirrigator, on an individual level. H. diversicolor buried a higher percentage (13%) of luminophores below the top 0.5 cm surface layer than M. neglecta (6%). D_b did not differ between the two species (2.4 × 10^− 3 cm² d^− 1) indicating a similar rate of diffusive mixing of the top sediment, however, the non-local transport parameter r was 2.5 y^− 1 for H. diversicolor and zero for M. neglecta, suggesting no significant particle-transport below the biodiffusive layer by M. neglecta. The average individual net bromide fluxes obtained were ca. 0.01 mL min^− 1 for H. diversicolor and 0.003 mL min^− 1 for M. neglecta, corresponding to an area-specific rate of ca. 12 L m^− 2 d^− 1 at the used densities. D_a did not differ between the two polychaetes, suggesting a higher individual solute exchange efficiency of M. neglecta considering the much higher ventilation rates reported for H. diversicolor than for Marenzelleria sp. The ongoing colonization of Baltic Sea sediments by M. neglecta at high densities may thus lead to an enhanced soluble release of both nutrients and contaminants. These results add information to the understanding of the potential effects of the invasion of M. neglecta on sediment biogeochemistry when competing with and/or replacing native species.     

Binding and direct electrochemistry of OmcA, an outer-membrane cytochrome from an iron reducing bacterium, with oxide electrodes: A candidate biofuel cell system

Dissimilatory iron-reducing bacteria transfer electrons to solid ferric respiratory electron acceptors. Outer-membrane cytochromes expressed by these organisms are of interest in both microbial fuel cells and biofuel cells. We use optical waveguide lightmode spectroscopy (OWLS) to show that OmcA, an 85 kDa decaheme outer-membrane c-type cytochrome from Shewanella oneidensis MR-1, adsorbs to isostructural Al₂O₃ and Fe₂O₃ in similar amounts. Adsorption is ionic-strength and pH dependent (peak adsorption at pH 6.5-7.0). The thickness of the OmcA layer on Al₂O₃ at pH 7.0 [5.8 ± 1.1 (2σ) nm] from OWLS is similar, within error, to that observed using atomic force microscopy (4.8 ± 2 nm). The highest adsorption density observed was 334 ng cm⁻² (2.4 × 10¹² molecules cm⁻²), corresponding to a monolayer of 9.9 nm diameter spheres or submonolayer coverage by smaller molecules. Direct electrochemistry of OmcA on Fe₂O₃ electrodes was observed using cyclic voltammetry, with cathodic peak potentials of −380 to −320 mV versus Ag/AgCl. Variations in the cathodic peak positions are speculatively attributed to redox-linked conformation change or changes in molecular orientation. OmcA can exchange electrons with ITO electrodes at higher current densities than with Fe₂O₃. Overall, OmcA can bind to and exchange electrons with several oxides, and thus its utility in fuel cells is not restricted to Fe₂O₃.     

Structural and equilibrium studies on mixed ligand complexes of Co(II), Ni(II), Cu(II) and Zn(II) with N-(2-benzimidazolyl)methyliminodiacetic acid and typical N, N donor ligands

Mixed ligand complexes: [Co(L)(bipy)] · 3H₂O (1), [Ni(L)(phen)] · H₂O (2), [Cu(L)(phen)] · 3H₂O (3) and [Zn(L)(bipy)] · 3H₂O (4), where L²⁻ = two -COOH deprotonated dianion of N-(2-benzimidazolyl)methyliminodiacetic acid (H₂bzimida, hereafter, H₂L), bipy = 2,2′ bipyridine and phen = 1,10-phenanthroline have been isolated and characterized by elemental analysis, spectral and magnetic measurements and thermal studies. Single crystal X-ray diffraction studies show octahedral geometry for 1, 2 and 4 and square pyramidal geometry for 3. Equilibrium studies in aqueous solution (ionic strength I = 10⁻¹ mol dm⁻³ (NaNO₃), at 25 ± 1 °C) using different molar proportions of M(II):H₂L:B, where M = Co, Ni, Cu and Zn and B = phen, bipy and en (ethylene diamine), however, provides evidence of formation of mononuclear and binuclear binary and mixed ligand complexes: M(L), M(H₋₁L)⁻, M(B)²⁺, M(L)(B), M(H₋₁L)(B)⁻, M₂(H₋₁L)(OH), (B)M(H₋₁L)M(B)⁺, where H₋₁L³⁻ represents two -COOH and the benzimidazole N₁-H deprotonated quadridentate (O⁻, N, O⁻, N), or, quinquedentate (O⁻, N, O⁻, N, N⁻) function of the coordinated ligand H₂L. Binuclear mixed ligand complex formation equilibria: M(L)(B) + M(B)²⁺ ? (B)M(H₋₁L)M(B)⁺ + H⁺ is favoured with higher π-acidity of the B ligands. For Co(II), Ni(II) and Cu(II), these equilibria are accompanied by blue shift of the electronic absorption maxima of M(II) ions, as a negatively charged bridging benzimidazolate moiety provides stronger ligand field than a neutral one. Solution stability of the mixed ligand complexes are in the expected order: Co(II) < Ni(II) < Cu(II) > Zn(II). The Δ log K_M values are less negetive than their statistical values, indicating favoured formation of the mixed ligand complexes over the binary ones.     

The base exchange reaction of NAD+ glycohydrolase: identification of novel heterocyclic alternative substrates

ADP-ribosyl cyclase and NAD⁺ glycohydrolase (CD38, E.C.3.2.2.5) efficiently catalyze the exchange of the nicotinamidyl moiety of NAD⁺, nicotinamide adenine dinucleotide phosphate (NADP⁺) or nicotinamide mononucleotide (NMN⁺) with an alternative base. 4′-Pyridinyl drugs (amrinone, milrinone, dismerinone and pinacidil) were efficient alternative substrates (k_cat/K_M = 0.9-10 μM⁻¹ s⁻¹) in the exchange reaction with ADP-ribosyl cyclase. When CD38 was used as a catalyst the k_cat/K_M values for the exchange reaction were reduced two or more orders of magnitude (0.015-0.15 μM⁻¹ s⁻¹). The products of this reaction were novel dinucleotides. The values of the equilibrium constants for dinucleotide formation were determined for several drugs. These enzymes also efficiently catalyze the formation of novel mononucleotides in an exchange reaction with NMN⁺, k_cat/K_M = 0.05-0.4 μM⁻¹ s⁻¹. The k_cat/K_M values for the exchange reaction with NMN⁺ were generally similar (0.04-0.12 μM⁻¹ s⁻¹) with CD38 and ADP-ribosyl cyclase as catalysts. Several novel heterocyclic alternative substrates were identified as 2-isoquinolines, 1,6-naphthyridines and tricyclic bases. The k_cat/K_M values for the exchange reaction with these substrates varied over five orders of magnitude and approached the limit of diffusion with 1,6-naphthyridines. The exchange reaction could be used to synthesize novel mononucleotides or to identify novel reversible inhibitors of CD38.     

Effect of thiocyanate on the peroxidase and pseudocatalase activities of Leishmania major ascorbate peroxidase

We report here that the Leishmania major ascorbate peroxidase (LmAPX), having similarity with plant ascorbate peroxidase, catalyzes the oxidation of suboptimal concentration of ascorbate to monodehydroascorbate (MDA) at physiological pH in the presence of added H₂O₂ with concurrent evolution of O₂. This pseudocatalatic degradation of H₂O₂ to O₂ is solely dependent on ascorbate and is blocked by a spin trap, α-phenyl-n-tert-butyl nitrone (PBN), indicating the involvement of free radical species in the reaction process. LmAPX thus appears to catalyze ascorbate oxidation by its peroxidase activity, first generating MDA and H₂O with subsequent regeneration of ascorbate by the reduction of MDA with H₂O₂ evolving O₂ through the intermediate formation of O₂⁻. Interestingly, both peroxidase and ascorbate-dependent pseudocatalatic activity of LmAPX are reversibly inhibited by SCN⁻ in a concentration dependent manner. Spectral studies indicate that ascorbate cannot reduce LmAPX compound II to the native enzyme in presence of SCN⁻. Further kinetic studies indicate that SCN⁻ itself is not oxidized by LmAPX but inhibits both ascorbate and guaiacol oxidation, which suggests that SCN⁻ blocks initial peroxidase activity with ascorbate rather than subsequent nonenzymatic pseudocatalatic degradation of H₂O₂ to O₂. Binding studies by optical difference spectroscopy indicate that SCN⁻ binds LmAPX (Kd = 100 ± 10 mM) near the heme edge. Thus, unlike mammalian peroxidases, SCN⁻ acts as an inhibitor for Leishmania peroxidase to block ascorbate oxidation and subsequent pseudocatalase activity.     

H2O2 and (.)NO scavenging by Mycobacterium leprae truncated hemoglobin O

Kinetics of ferric Mycobacterium leprae truncated hemoglobin O (trHbOFe(III)) oxidation by H₂O₂ and of trHbOFe(IV)O reduction by NO and NO₂⁻ are reported. The value of the second-order rate constant for H₂O₂-mediated oxidation of trHbOFe(III) is 2.4 × 10³ M⁻¹ s⁻¹. The value of the second-order rate constant for NO-mediated reduction of trHbOFe(IV)O is 7.8 × 10⁶ M⁻¹ s⁻¹. The value of the first-order rate constant for trHbOFe(III)ONO decay to the resting form trHbOFe(III) is 2.1 × 10¹ s⁻¹. The value of the second-order rate constant for NO₂⁻-mediated reduction of trHbOFe(IV)O is 3.1 × 10³ M⁻¹ s⁻¹. As a whole, trHbOFe(IV)O, generated upon reaction with H₂O₂, catalyzes NO reduction to NO₂⁻. In turn, NO and NO₂⁻ act as antioxidants of trHbOFe(IV)O, which could be responsible for the oxidative damage of the mycobacterium. Therefore, Mycobacterium leprae trHbO could be involved in both H₂O₂ and NO scavenging, protecting from nitrosative and oxidative stress, and sustaining mycobacterial respiration.     

Dissimilatory perchlorate reduction: a review

In the United States anthropogenic activities are mainly responsible for the wide spread perchlorate contamination of drinking water, surface water, groundwater, and soil. Even at microgram levels, perchlorate causes toxicity to flora and fauna and affects growth, metabolism and reproduction in humans and animals. Reports of antithyroid effects of perchlorate and its detection in common food items have raised serious public health concerns, leading to extensive decontamination efforts in recent years. Several physico-chemical removal and biological decontamination processes are being developed. Although promising, ion exchange is a non-selective and incomplete process as it merely transfers perchlorate from water to the resin. The perchlorate-laden spent resins (perchlorate 200-500 mg L⁻¹) require regeneration resulting in production of concentrated brine (6-12% NaCl) or caustic waste streams. On the contrary, biological reduction completely degrades perchlorate into O₂ and innocuous Cl⁻. High reduction potential of ClO₄⁻/Cl⁻ (E° = ∼1.28 V) and ClO₃⁻/Cl⁻ pairs (E° = 1.03 V) makes these contaminants thermodynamically ideal e⁻ acceptors for microbial reduction. In recent years unique dissimilatory perchlorate reducing bacteria have been isolated and detailed studies pertaining to their microbiological, biochemical, genetics and phylogenetic aspects have been undertaken which is the subject of this review article while the various physico-chemical removal and biological reduction processes have been reviewed by others.